The calcium and voltage regulated BK-type K+ channel encoded by the Slo1 gene is a widely expressed ion channel impacting on regulation of excitability in a variety of tissues. Diversity in function of the BK channel arises from tissue-specific expression of up to four different auxiliary b subunits (b1-b4) and a newly identified family of g subunits. b1 and b4 subunits have been implicated in hypertension and epilepsy, respectively, and other indications suggest that BK channels may be therapeutic targets in stroke, hypertension, epilepsy, and tumor growth regulation. Of auxiliary subunits, little is known about physiological roles of b2 and b3 subunits, both of which produce use- dependent changes in BK currents and even less is known about g subunits. In this project, mechanisms of use-dependent regulation of BK currents by b2 and b3 subunits will be examined. Furthermore, the consequences of assembly of multiple kinds of auxiliary (both b and g) subunits into single channels will be tested and the rules governing b and g subunit coassembly in BK channels determined. This project is expected to provide mechanistic and physiological insight into the role of two major regulators of BK channels, the b2 and b3 subunits and new insight into the role of g subunits.